Structure of the
bikunin. The portion on the left corresponds to
the sugar part of the molecule, the sequence of which was
determined in the current study. The portion on the right
corresponds to the protein part of bikunin.

DNA and protein sequencing have forever transformed science,
medicine, and society. Understanding the structure of these
complex biomolecules has revolutionized drug development,
medical diagnostics, forensic science, and our understanding of
evolution and development. But, one major molecule in the
biological triumvirate has remained largely uncharted:
carbohydrate biopolymers.

Today, for the first time ever, a team of researchers led by
Robert Linhardt of Rensselaer Polytechnic Institute has
announced in the October 9 Advanced Online Publication edition
of the journal Nature Chemical Biology the sequence of
a complete complex carbohydrate biopolymer. The surprising
discovery provides the scientific and medical communities with
an important and fundamental new view of these vital
biomolecules, which play a role in everything from cell
structure and development to disease pathology and blood
clotting.

The paper is titled “The proteoglycan bikunin has a defined
sequence.”

“Carbohydrate biopolymers, known as glycosaminoglycans,
appear to be really important in how cells interact in higher
organisms and could explain evolutionary differences and how
development is driven. We also know that carbohydrate chains
respond to disease, injury, and changes in the environment,”
said Linhardt, who is the Ann and John H. Broadbent Jr. ’59
Senior Constellation Professor of Biocatalysis and Metabolic
Engineering at Rensselaer. “In order to understand how and why
this all happens, we first need to know their structure. And
today, at least for the simplest glycosaminoglycan structure,
we can now do this.”

The first glycosaminoglycan sequenced was obtained from
bikunin. Bikunin is a proteoglycan, a protein to which a single
glycosaminoglycan chain is attached. Unlike less sophisticated
carbohydrate biopolymers, such as starch and cellulose, the
proteoglycans are decorated with structurally complex
carbohydrates that enable them to perform more sophisticated
and defined roles in the body. Bikunin, for example, is a
natural anti-inflammatory that is used as a drug for the
treatment of acute pancreatitis in Japan. It has the simplest
chemical structure of any proteoglycan. Linhardt views the
discovery of the structure of bikuin as the first step on the
ladder to the discovery of the structure of more complex
proteoglycans.

“The first genome sequences of DNA were on the simplest
organisms such as bacteria. Once the technology was developed
it ultimately led to the sequencing of the human genome,” he
said. “In our efforts to sequence carbohydrate biopolymers we
don’t yet know if the defined structure we observe for this
simple protoglycan will hold for much more complex
proteoglycans.” But, looking for structure in more complex
proteoglycans will be among the next steps in the research for
Linhardt and his team. The search for structure could help put
to rest a long-running debate in the scientific community as to
whether complex carbohydrate biopolymers require a defined
structure to function.

“Despite all that is known about glycan formation, our
understanding has not yet been deep enough to infer sequence or
even determine if sequence occurs,” Linhardt said. “These
findings represent a new way of looking at these complex
biomolecules as ordered structures.”

Linhardt’s research into carbohydrate sequencing began 30
years ago. In his previous work, he determined that some order
existed in at least a portion of some carbohydrate biopolymers,
but it did not represent the entire finished puzzle.

“Previously, we could see a pattern, but we could not see if
all the chains were playing the same music. The tools did not
yet exist. Now we can recognize it as a symphony.”

To uncover the entire structure, Linhardt and his team,
which was led by his doctoral student Mellisa Ly, borrowed a
technique from the field of protein research called the
proteomics top-down approach. As opposed to the bottom-up
approach that first breaks apart a complex biopolymer into
pieces and then rebuilds it piece by piece like a jigsaw
puzzle, the top-down approach used by Linhardt and colleagues
allows the researcher to picture the whole intact puzzle. This
can only be accomplished with some of the most sophisticated
technology available to the scientific community today,
including very high-powered mass spectrometers.

Linhardt used a mass spectrometer located in the Rensselaer
Center for Biotechnology and Interdisciplinary Studies (CBIS)
to make his initial discoveries, and had these results
independently confirmed on a separate and higher-level
spectrometer at the University of Georgia. Mass spectrometers
break down a molecule into separate charged particles or ions.
These ions can then be categorized and analyzed based on their
mass-to-charge ratio. These ratios then allow for sequencing of
the entire molecule.

“This was truly the convergence of really sophisticated
spectroscopy and its application to biology,” Linhardt said.
“We were fortunate to have a lot of time to play with the
instrument at CBIS to understand its capabilities.”

Beyond the technology it also took faith and determination.
According to Linhardt, “It takes a student that is willing to
try something even when the odds are pretty low. If it doesn’t
work, you make incremental progress. If it does work, you can
make a great discovery. But, from the beginning you need to be
a believer that it is worth taking the chance because it takes
a lot of hard work in the lab.”

And the odds weren’t in Linhardt’s favor. Despite being the
most simple of proteoglycans, there were still 290 billion
different possible sequences for the molecule.

“The first sample we looked at, we got the structure,”
Linhardt said. “In the end we did 15 chains and they all came
back playing the same exact symphony.”

The research is funded by the National Institutes of
Health.

Linhardt and Ly were joined in the research by Tatiana
Laremore of Rensselaer; Franklin Leach and Jonathan Amster of
the University of Georgia; and Toshihiko Toida of Chiba
University in Japan.